Stretch-induced alternative splicing of serumresponse factor promotes bronchialmyogenesis and is defective in lung hypoplasia

Yan Yang, … , Ilana Ariel, Lucia Schuger

J Clin Invest. 2000;106(11):1321-1330. https://doi.org/10.1172/JCI8893.

Smooth muscle (SM) develops only in organs and sites that sustain mechanical tensions.Therefore, we determined the role of stretch in mouse and human bronchial myogenesis.Sustained stretch induced expression of SM proteins in undifferentiated mesenchymal cellsand accelerated the differentiation of cells undergoing myogenesis. Moreover, bronchialmyogenesis was entirely controlled in lung organ cultures by the airway intraluminalpressure. Serum response factor (SRF) is a transcription factor critical for the induction ofmuscle-specific gene expression. Recently, a SRF-truncated isoform produced byalternative splicing of exon 5 has been identified (SRFΔ5). Here we show thatundifferentiated mesenchymal cells synthesize both SRF and SRFΔ5 but that SRFΔ5synthesis is suppressed during bronchial myogenesis in favor of increased SRF production.Stretch induces the same change in SRF alternative splicing, and its myogenic effect isabrogated by overexpressing SRFΔ5. Furthermore, human hypoplastic lungs related toconditions that hinder cell stretching continue to synthesize SRFΔ5 and show a markeddecrease in bronchial and interstitial SM cells and their ECM product, tropoelastin. Takentogether, our findings indicate that stretch plays a critical role in SM myogenesis andsuggest that its decrease precludes normal bronchial muscle development.

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IntroductionVisceral smooth muscle (SM) originates from localmesenchymal cells that in early-midgestation begin tosynthesize SM proteins, including SM α-actin, desmin,SM myosin, SM22, and calponin in a specific periair-way distribution (1–5). In the mouse developing respi-ratory system, cells expressing SM proteins are firstdetected in the trachea on day 11 of gestation (3, 4),and then SM differentiation proceeds in a cranial-to-caudal fashion to form the bronchial musculature(1–5). The other type of visceral SM cells found in thelung are interstitial SM cells, also known as interstitialcontractile cells, or myofibroblasts. Interstitial SM cellsare originally located at the sites of future alveolar sep-tae, and, in the mature organ, they form part of the sep-tae tips (6). Except for the aorta, the development of thevascular musculature lags behind that of visceral SMby several days (4, 7–9).

Unlike striated muscle differentiation, on which con-siderable information was gathered over the years, themechanisms and genetic program that control SMmyogenesis remain, for the most part, unknown. Weand others have observed that lung mesenchymal cellprecursors change their shape from round to elongat-ed before undergoing bronchial SM differentiation (ref.3; Y. Yang and L. Schuger, unpublished observations).Based on this observation we recently examined

whether changes in cell shape might play a role in air-way myogenesis. Unexpectedly, our studies demon-strated that essentially all undifferentiated embryonicmesenchymal cells are potential SM precursors(10–12). These studies also confirmed the critical roleof cell shape in myogenesis. Specifically, we found thatcell rounding prezvents myogenesis, regardless of thenormal fate of the cell in vivo, whereas cell spread-ing/elongation induces SM differentiation, even inmesenchymal cells from nonmuscular organs (10–12).

Developing tubular tissues, such as those of the res-piratory, gastrointestinal, and urinary systems, arefilled with liquid. As a consequence, the periluminalmesenchymal cells are subjected to mechanical ten-sion/stretch exerted by the liquid’s hydrostatic pressure(13). These forces likely represent a significant factor indetermining the periluminal mesenchymal cell shape.In the developing lung, cells are additionally subjectedto repeated stretch caused by intrauterine breathing(13). The fact that mechanical stretch causes cell elon-gation and that cell elongation is likely to be sensed bythe cell as a mechanical stimulus suggested to us thatcell tension/stretch may play an important role in theprocess of visceral myogenesis.

Here we used a combination of lung cell and organ cul-tures from fetal mouse and human origin to determinethe effect of mechanical stretch upon SM myogenesis.

The Journal of Clinical Investigation | December 2000 | Volume 106 | Number 11 1321

Stretch-induced alternative splicing of serum responsefactor promotes bronchial myogenesis and is defective in lung hypoplasia

Yan Yang,1 Safedin Beqaj,1 Paul Kemp,2 Ilana Ariel,3 and Lucia Schuger1

1Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan, USA2Department of Biochemistry, Cambridge University, Cambridge, United Kingdom3Department of Pathology, Haddash University Hospital, Hebrew University School of Medicine, Jerusalem, Israel

Address correspondence to: Lucia Schuger, Department of Pathology, Wayne State University, 540 E. Canfield, Detroit, Michigan 48201, USA. Phone: (313) 577-5651; Fax: (313) 577-0057; lschuger@med.wayne.edu.

Received for publication November 11, 1999, and accepted in revised form October 26, 2000.

Smooth muscle (SM) develops only in organs and sites that sustain mechanical tensions. Therefore,we determined the role of stretch in mouse and human bronchial myogenesis. Sustained stretchinduced expression of SM proteins in undifferentiated mesenchymal cells and accelerated the dif-ferentiation of cells undergoing myogenesis. Moreover, bronchial myogenesis was entirely controlledin lung organ cultures by the airway intraluminal pressure. Serum response factor (SRF) is a tran-scription factor critical for the induction of muscle-specific gene expression. Recently, a SRF-trun-cated isoform produced by alternative splicing of exon 5 has been identified (SRF∆5). Here we showthat undifferentiated mesenchymal cells synthesize both SRF and SRF∆5 but that SRF∆5 synthesisis suppressed during bronchial myogenesis in favor of increased SRF production. Stretch induces thesame change in SRF alternative splicing, and its myogenic effect is abrogated by overexpressingSRF∆5. Furthermore, human hypoplastic lungs related to conditions that hinder cell stretching con-tinue to synthesize SRF∆5 and show a marked decrease in bronchial and interstitial SM cells and theirECM product, tropoelastin. Taken together, our findings indicate that stretch plays a critical role inSM myogenesis and suggest that its decrease precludes normal bronchial muscle development.

J. Clin. Invest. 106:1321–1330 (2000).

We found that sustained stretch was by itself sufficientto induce expression of SM proteins in lung undifferen-tiated mesenchymal cells and to accelerate synthesis ofSM proteins in mesenchymal cells already undergoingmyogenic differentiation. Conversely, SM myogenesisdid not take place in the absence of mechanical stimuli.

Many transcription factors have been linked to thecontrol of SM-specific gene expression. Among them,one of the best studied is serum response factor (SRF)(14–16), a member of the MADS (MCM-1, agamous anddeficiens, and SRF) box family of transcription factors.SRF binds to the CArG box or CArG box–like motif, anessential cis-element present in muscle-specific proteinssuch as SM α-actin, SM22, SM myosin, β-tropomyosin,and caldesmon, and stimulates their transcription.

Although SRF has been studied for many years, onlyrecently has it been demonstrated that several trun-cated isoforms are produced by alternative splicingfrom the same SRF pre-mRNA (17, 18). The newlyidentified SRF isoforms are SRF∆5 (17), also referredto as SRF-M (18), lacking exon 5, SRF-S lacking exons4 and 5, and SRF-I lacking exons 3, 4, and 5. All theseSRF species lack regions of the COOH-terminal trans-activation domain but have intact DNA-bindingdomains located in the NH2 terminus. Here we showthat stretch induces myogenic differentiation by sup-pressing SRF∆5 in SM cell precursors in favor ofincreased SRF production.

Finally, our studies demonstrate that humanhypoplastic lungs related to conditions that diminishlung distention exhibit a severe decrease in visceral mus-cle and elastin and continue to synthesize SRF∆5. Thesenew findings underscore the importance of mechanicalstimuli in the control of visceral myogenesis.

MethodsCell-stretching device. A cell-stretching device was manu-factured according to our specifications by MichaelMonford (Department of Bioengineering, Universityof California at San Diego, San Diego, California,USA). It consisted of a rectangular acrylic dish with asilicone membrane (Silastic; Specialty ManufacturingInc., Saginaw, Michigan, USA) fitted to the bottomand attached to removable clamps (Figure 1a). Thecells were plated on the membrane coated with 0.1%poly-L-lysine to maintain the cells in an undifferenti-ated state (round in shape) or 0.01% poly-L-lysine orcollagen I (10 µg/cm2) to allow spread-induced SM dif-ferentiation (10, 12). Static axial-stretching forces wereapplied after cell attachment was complete by placingthe clamps into different slots (Figure 1a). The mag-nitude of stretching was represented as the percentageof membrane distention from its original length to itslength after stretching.

Cells. Crl: CD-1 (ICR) BR mice (Charles River, Wilm-ington, Massachusetts, USA) were mated, and the daya vaginal plug was identified was designated as day 0 ofembryonic development. Undifferentiated mesenchy-mal cells were isolated by differential plating (19) from

embryonic intestine and lung on day 11 and from kid-ney on day 12 of gestation. Absence of SM proteins inthese organs at this stage was confirmed by previousstudies (11). Human lung embryonic mesenchymalcells were also obtained from voluntary pregnancy ter-minations (see below) by differential plating. The cellswere isolated directly on the silicone membranes andcultured in MEM with 10% FBS (Irvine Scientific,Santa Ana, California, USA) for up to 24–48 hours. Toobtain fully differentiated SM cells (SM protein syn-thesis and electrical responses comparable to those ofmature SM cells), the mesenchymal cells were culturedfor 96 hours under conditions that allow cell spreading(11) before stretching and/or transfection. In someexperiments, cell proliferation was prevented by reduc-ing the FBS to 1% (10–11).

Lung organ culture. Lungs were microdissected fromday 11 CD-1 mice and cultured embedded in a thin col-lagen gel (5 milligrams collagen I per milliliter of medi-um) layered on a transwell device (Figure 1b). Lung sam-ples from ten voluntary pregnancy terminations (16–18weeks) were obtained from Advanced BioscienceResources Inc. (ABR, Alameda, California, USA). Frag-ments of peripheral lung parenchyma (including ter-minal sacs and surrounding mesenchyme) ranging insize from 1 to 4 mm3 were microdissected and culturedas described for the mouse lung. The airway intralumi-nal fluid was microaspirated from the organ culturesand replaced with a solution of MEM-10% FBS and var-ious concentrations of dextran (Life Technologies Inc.,Grand Island, New York, USA). Dextran is 68-kDa poly-saccharide used clinically as an osmotic volumeexpander. Here we used dextran to increase the airwayintraluminal pressure and thereby stretch the sur-rounding mesenchymal cells. Alternatively, dextran wasdissolved in the culture medium surrounding the tis-sues to accomplish the opposite effect (Figure 1b).Explants were cultured submerged in serumless medi-um (BGjb; Life Technologies Inc.) for 24 hours. At theend of the culture period, the lungs were lysed or fixedas a whole mount for SM-specific protein detection. Insome experiments, the main airways, including the peri-bronchial mesenchyme, were microdissected from freshor cultured murine lungs and trypsinized. The peri-bronchial mesenchymal cells were then further selectedby 45 minutes’ differential plating (19) and immediate-ly lysed for SRF isoform evaluation.

Measurement of stretch-induced cell elongation. Round-cellcultures were stretched by 0%, 1%, 5%, and 10% of thetotal silastic membrane length and fixed in 1%paraformaldehyde immediately and after 1, 2, and 6hours of stretching. The airway intraluminal fluid ofmouse lung organ cultures was replaced with 0%, 1%,5%, 10%, or 15% dextran in BGjb or 1% dextran wasadded to the culture medium outside the lung. Prelim-inary studies showed that dextran concentrations high-er than 2.5% in the culture medium cause osmoticdamage. The tissues were frozen after 4 and 24 hoursin culture. Preliminary studies indicated that the

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increment in peribronchial mesenchymal cell diameterwas fully elicited 4 hours after dextran microinjectionand remained constant for up to 24 hours in culture.Five micrometer–thick sections were cut and stainedwith hematoxylin and eosin for light-microscopicobservation. Stretch-induced cell elongation was deter-mined on computer-scanned images. In cell cultures,the cell diameter for each stretching force was meas-ured in 50 cells per treatment. In lung organ cultures(in which the cell boundaries are not defined), the dis-tance between the centers of adjacent peribronchialmesenchymal cell nuclei was measured in the distalbronchial buds. Increments in this distance were con-sidered a measure of cell stretch. At least 40 distancesper treatment were recorded.

Human hypoplastic lungs. Formalin-fixed, paraffin-embedded tissue samples from nine hypoplastic lungsand nine gestational age–matched controls ranging ingestational age from 20 to 25 weeks were selected fromautopsy files. Four hypoplastic lungs (two female, twomale) were related to oligohydramnion due to renalagenesis and five (two female, three male) to leftdiaphragmatic hernia. The lungs had a 35–60% reduc-tion in their expected weight, the most extreme reduc-tion found in the lungs isolateral to a diaphragmatichernia and the less severe in lungs contralateral to it.None of the neonates survived for more than a fewminutes after delivery.

Immunoblot analysis. The following Ab’s were usedfor detection of SM-specific proteins: a mouse mAb toSM α-actin (Boehringer Mannheim Biochemicals Inc.,Indianapolis, Indiana, USA) at a concentration of 0.25 µg/ml, a mouse mAb to desmin (DAKO Corp.,Carpinteria, California, USA) at a concentration of1.125 µg/ml, rabbit polyclonal Ab’s to SM-myosin(Biomedical Technologies, Stoughton, Massachusetts,USA) at a concentration of 10 µg/ml, and rabbit poly-clonal Ab’s to SM22 (gift from Rodrigo Bravo, Bristol-Myers Squibb Pharmaceutical Research Institute,Princeton, New Jersey, USA) at a concentration of 2 µg/ml. A rabbit polyclonal Ab against mousetropoelastin and another against human tropoelastin(Elastin Products, St. Louis, Missouri, USA) were usedat a dilution of 1:000. Rabbit polyclonal Ab againstSRF/SRF∆5 (Santa Cruz Biotechnology, Santa Cruz,California, USA) was used at a 1:200 dilution. Cell cul-tures were lysed, and immunoblots were performed asdescribed previously (10, 11).

Immunohistochemistry. Five micrometer–thick sectionsfrom formalin-fixed human lung explants, hypoplas-tic lungs, and matched controls were immunostainedwith Ab’s against SM α-actin. Hypoplastic lungs andtheir controls were additionally stained with Ab’sagainst tropoelastin, low-molecular-weight cytoker-atins (rabbit polyclonal Ab from DAKO Corp.) andPECAM-1 (mouse monoclonal Ab from DAKO Corp.).Ab’s to SM α-actin and PECAM-1 were used at a con-centration of 1 µg/ml. Ab’s to cytokeratins and tropoe-lastin were used at a dilution of 1:200. Mouse embry-

onic lung explants were immunostained as wholemounts (without sectioning). Staining was completedusing commercial peroxidase–anti-peroxidase kits(DAKO Corp.) or FITC-conjugated secondary Ab’s asdescribed previously (10, 20).

RNA isolation. RNA was isolated from cells and tissueswith TRIzol reagent (Life Technologies Inc.) followingthe manufacturer’s instructions. To isolate RNA fromformalin-fixed, paraffin-embedded lungs, six 20-µm-thick sections were cut, collected in Eppendorf tubes,and dewaxed using two changes of xylene for 10 min-utes each at 60°C. The samples were dehydrated indecreasing concentrations of alcohol (100%, 70%, 50%)for 10 minutes each and dried in a Speed Vac for 3–4minutes. Lysis buffer was added to the tubes (20 mMTris, pH 7.5, 20 mM EDTA, 2.5 µg/ml proteinase K, 1%SDS), and these were incubated overnight at 55°C. Thesamples were then spun down, the buffer was removed,and RNA was extracted with TRIzol.

RT-PCR. The following primers were used for PCR: SMα-actin: 5′ forward primer, 5′-TCCCTGGAGAAGAGC-TACGA-3′, and 3′ reverse primer, 5′-GGGCTTTTAATCTC-CTTCGG-3′. Desmin: 5′ forward primer, 5′-GTGAA-GATGGCCTTGGATGT-3′, and 3′ reverse primer,5′-GTAGCCTCGCTGACAACCTC-3′. SM22: 5′ forwardprimer, 5′-TGTGACCAAAAACGATGGAA-3′, and 3′ reverseprimer, 5′-ATAGGCATTTGTGAGGCAGG-3′. SM-myosin:5′ forward primer, 5-GACAACTCCTCTCGCTTTGG-3′,and 3′ reverse primer, 5′-GCTCTCCAAAAGCAGGTCAC-3′.Tropoelastin: 5′ forward primer, 5′-AGATGGCTCCTCA-CACTGGT-3′, and 3′ reverse primer, 5′-AGCACCACCAC-CTGGATAAA-3′. SRF isoforms: 5′ forward primer, 5′-ATCACCAACTACCTGGCACC-3′, and 3′ reverse primer,5′-CACCTGTAGCTCGGTGAGGT-3′. The same set ofprimers was used to amplify both SRF isoforms in mice

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Figure 1(a) The cell-stretching device consists of a rectangular acrylic dish witha stretchable silastic membrane fitted to the bottom and attached toremovable clamps. The embryonic mesenchymal cells are cultured onthe membrane coated with 0.1% poly-L-lysine to maintain the cells’round shape and thereby prevent spread-induced SM differentiationor on 0.01% poly-L-lysine or collagen I to allow spread-induced SMdifferentiation. The membrane is longitudinally stretched up to sev-eral lengths by placing the clamps into different slots (labeled 1 to 6).(b) Diagram of the organ culture system used in this study is shown.Mouse and human embryonic lung explants are cultured embeddedin a thin collagen gel layered on a transwell device. Dextran, a volumeexpander polysaccharide, is microinjected into the airways to increasethe intraluminal pressure by attracting liquid. Alternatively, dextran isdissolved in the culture medium to obtain the opposite effect.

and humans. RT-PCR was performed with theGeneAmp RNA PCR kit (Perkin-Elmer Applied Biosys-tems, Foster City, California, USA), as described previ-ously (11). Twenty cycles were run for all amplificationsbesides tropoelastin, which was run for 28 cycles, andSRF, which was run for 20 to 35 cycles. Message for allthe SM-specific proteins, except SM myosin, was detect-ed in the round cells if the number of cycles wasincreased above 30. SM myosin mRNA was not detect-ed in round cells even after 50 cycles.

SRF and SRF∆5 transfections. SRF and SRF∆5 cDNAscloned into pGEM-T easy (Promega Corp., Madison,Wisconsin, USA) (18) were released from the vector byEcoRI and ligated into EcoRI-digested pCDNA3 expres-sion vector (Invitrogen Corp., Carlsbad, California,USA). The orientation of the clones was determined byrestriction digestion with PstI, and the sequence wasconfirmed. Primary cultures of mouse lung embryonicmesenchymal cells were transfected 1 hour after attach-ment was completed using Lipofectamine plus reagent(Life Technologies Inc.), following the manufacturer’sinstructions. The plasmids and empty vector were mixedwith the Lipofectamine reagent in a 1:3.5 wt/vol pro-portion, and the cells were transfected for 3 hours in thepresence of 10% FBS. In the experiments in which thecells were stretched, transfection and stretching were ini-tiated concomitantly. In this case the cells were allowedto spread since, in our experience, round cells are diffi-cult to transfect. The cells were lysed 18 hours aftertransfection. This experimental approach does not allowfor determination of transfection efficiency. However,studies using hemagglutinin-tagged plasmid constructsunrelated to SRF indicated that approximately 30–35%of the cells in our primary cultures are transfected (S.Beqaj and L. Schuger, unpublished observation).

To determine whether transfections affected total pro-tein synthesis, control and transfected cells were meta-bolically labeled with 100 µCi/well of [35S]-methionine(NEN-Dupont, Boston, Massachusetts, USA). [35S]-methionine was added to the cultures concomitantlywith the plasmid constructs. At the end of the cultureperiod, the cells were lysed, and the protein was precip-itated with 10% trichloroacetic acid. The [35S]-methion-ine incorporated into 1 µg of precipitated protein wasdetermined as cpm in a β-scintillator counter.

ResultsStretch induced mouse-lung undifferentiated mesenchymal cellsto express SM proteins and stimulated differentiation of SMmyoblasts. In the first set of studies mesenchymal cellswere prevented from spreading (and undergoingspread-induced SM differentiation) by plating on 0.1%poly-L-lysine. Upon stretching, the cells increased theirmaximal diameter from 16 ± 2.5% with 1% stretching toup to 38 ± 1.6% with 10% membrane stretching (Figure2a). Lung undifferentiated mesenchymal cells respond-ed to 2 hours of 5% and 10% uniaxial stretching by turn-ing on the expression of SM myosin and drasticallyincreasing mRNA levels for SM α-actin, desmin, andSM22 (Figure 2, b and d). In these cells SM-specific pro-tein translation was initiated after 18 hours of stretch(Figure 2c). Immunohistochemical studies indicatedthat all of the cells in culture synthesized equal levels ofSM protein (not shown). Control (nonstretched) cellsand cells exposed to 1% stretch remained undifferenti-ated for the whole period studied (Figure 2, b and c) andsynthesized high levels of α-fetoprotein, an embryonicmarker (not shown). Stretching forces of 15% or higherdid not induce SM myosin expression and decreasedSM α-actin, desmin, and SM22 to levels undetectable by

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Figure 2(a) Measurement of mesenchymal cell elongation caused by dif-ferent uniaxial stretching forces. Undifferentiated (round) cellswere measured in this study. Each column represents the meanmaximal diameter of 50 cells ± SD. (b) RT-PCR shows inductionof SM-specific mRNA in undifferentiated embryonic lung mes-enchymal cells after 2 hours of 5% uniaxial stretch. (c)Immunoblot shows SM α-actin translation in embryonic lungmesenchymal cells after 18 hours of uniaxial stretch. (d) Lack ofmyogenic response to stretch in undifferentiated mesenchymalcells from the intestine and kidney after 2 hours of uniaxialstretch are shown. This study was done under conditions thatinhibit cell proliferation (1% FBS). (e) Immunoblots showstretch-induced upregulation of SM-specific protein synthesis inembryonic mesenchymal cells undergoing spread-induced myo-genic differentiation. Notice that desmin requires more time tobe stimulated. The cells are plated under conditions that allowcell spreading and then stretched for 2, 6, or 24 hours (lanes 2and 3). The nonstretched controls (lane 1) were cultured for 24hours. (f) Upon reaching full SM differentiation (96 hours in cul-ture, SM-specific protein levels and electrical properties ofmature SM cells; ref. 11), mesenchymal cells stop responding tostretch under the conditions tested here. The studies presentedin b–f were repeated more than three times with similar results.

RT-PCR (not shown). Mesenchymal cells from kidneyand intestine subjected to 1–15% stretching forcesremained undifferentiated (Figure 2d). These cells, how-ever, differentiated into SM cells upon spreading (11).The response to stretching was observed irrespective ofwhether the cells were cultured under conditions thatpromoted or inhibited cell proliferation, as indicated bythe expression of SM markers in mesenchymal cells cul-tured in 1% FBS (Figure 2d). Lung mesenchymal cellsundergoing spread-induced SM differentiation (platedon 0.01% poly-L-lysine or collagen I) responded tomechanical stretch by further upregulating the pro-duction of SM proteins (Figure 2e). Differentiated SMcells (after 96 hours in culture) showed no response touniaxial stretching (Figure 2f).

Stretch induced SM myogenesis in mouse embryonic lungorgan cultures. In mouse embryonic lung organ cultures,maximal diameter of distal peribronchial mesenchy-mal cells increased from 10 ± 6 µm (with no dextran)to 26 ± 8 µm with 15% intraluminal dextran (Figure 3a)

(in vivo normal SM cell diameter is 20 ± 10 µm).Although the magnitude of stretch and the dextranconcentration are graded similarly (1–15%), notice thatthese are not equivalent. For instance, 5% dextrancaused more cell elongation than 10% stretch. Also,unstretched cells have a slightly smaller diameter inexplants than in cell culture. In the absence of intralu-minal dextran, the lung explants developed somebronchial muscle after 24 hours in culture (Figure 3, band c). This may reflect the secretion of fluid by theepithelial cells (21), which is likely to cause some intra-luminal hydrostatic pressure. By comparison, a promi-nent bronchial SM layer was developed in response tothe stretch generated by 1–5% dextran (approximately20–60% cell elongation) (Figure 3, b and d). Dextranconcentrations higher than 15% resulted in less SMprotein synthesis (not shown). When intraluminalpressure was maintained at a minimum by increasingthe osmotic pressure outside the lung, no bronchialSM development was observed (Figure 3, b and e).

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Figure 3 (a) Determination of periluminal mesenchymal cell elongationcaused by different concentrations of dextran within the airways.Since mesenchymal cell boundaries are indistinguishable in vivo, wemeasured the distance between the centers of adjacent nuclei (themore the stretch, the more the distance). Each column representsthe mean measurement of 40 distances ± SD. (b) Immunoblotsshow modulation of SM protein synthesis in embryonic lung organcultures by dextran-induced peribronchial cell stretch. A dose-response increase in SM protein synthesis is seen in day-11 lungexplants containing dextran inside the airways after 24 hours in cul-ture (lanes 3–5). No SM protein synthesis is observed when dextranis present in the culture medium outside the lung, preventing devel-opment of intraluminal pressure (lane 2). Minimal synthesis of SMproteins is seen in the control lung explants in which some hydro-static pressure is likely to be produced by the fluid secreted byepithelial cells (lane 1). (c–e) Immunofluorescence shows modula-tion of bronchial myogenesis in embryonic lung organ cultures bydextran-induced peribronchial cell stretching. Day-11 lung explantswere cultured for 24 hours and then immunostained for SM α-actin as whole-mount preparations. (c) SM α-actin in lung explants culturedwithout dextran. Notice the occurrence of some bronchial SM development (arrows). (d) SM α-actin in lung explants cultured with 5% dex-tran inside the airways, where significant bronchial myogenesis took place (arrows). (e) In lung explants cultured in the presence of 1% dex-tran in the culture medium, notice the absence of bronchial SM development. Results shown in b–e are representative of three experiments,each performed on duplicate lung explants per treatment. Bar, 100 µm.

Figure 4(a) Immunoblot shows stretch-induced upregulation of SM-specificprotein synthesis in human lung mesenchymal cells undergoing myo-genic differentiation. (b) Immunoblots show stimulation of SM proteinsynthesis in human fetal lung organ cultures by dextran-induced tissuestretching. In a and b the lungs were obtained from 18-week fetuses,after the onset of visceral and vascular SM differentiation. Therefore,the initial levels of SM protein are higher than in murine cells. (c and d)Immunohistochemistry shows SM α-actin in histological sections fromhuman fetal lungs (18 weeks) cultured for 48 hours with 1% dextran inthe culture medium outside the explant (c) and 1% dextran inside theairways (d). Notice the significant increment in bronchial SM cells in ccompared with d (arrows). The vascular SM shows no differences(arrowheads). Results are representative of three experiments, each per-formed on quadruplicate lung explants per treatment. Bar, 20 µm.

Stretch leading to approximately 60% cell elongationstimulated myogenesis in lung explants, but inhibitedSM differentiation in cell cultures. These differencesare likely to be related to the more artificial conditionscreated by cell culture compared with organ culture.

Stretch stimulated the expression of SM proteins in humanlung embryonic mesenchymal cells and organ cultures.Human differentiating SM cells in primary culturesincreased their expression of SM proteins upon stretch-ing in a manner similar to that of mouse SM-differen-tiating cells (Figure 4a). Similarly, human lung explantsresponded to the stretch generated by 1–10% dextran(approximately 20–60% cell elongation) by increasingthe synthesis of SM proteins (Figures 4, b–d). Concen-trations greater than 15% resulted in less SM proteinsynthesis. The observation that SM markers are higherin unstretched human cells, compared with the murinecounterparts, likely reflects the more advanced devel-opmental stage of the human lungs and the presenceof some differentiated SM cells before organ culture.

Stretch regulated the synthesis of SRF and SRF∆5 in lungembryonic mesenchymal cell and organ cultures of mouse andhuman origin. Immunoblot analysis of peribronchial

mesenchymal tissue microdissected from mouse lungsbefore the onset of myogenesis (day 11) and lysed afterbrief plating (to separate epithelial cells) demonstratedthat these cells synthesize both SRF and SRF∆5 iso-forms in vivo (Figure 5a). However, bronchial SM cellsisolated in a similar manner from day 14 lungs (afterthe onset of bronchial muscle) synthesize only SRF(Figure 5a). Furthermore, levels of SRF message andprotein were higher in SM cells compared with theirundifferentiated counterparts, consistent with achange in SRF pre-mRNA alternative splicing. Sinceundifferentiated embryonic mesenchymal cells under-go myogenic differentiation upon spreading/elonga-tion in culture (10, 11), these were evaluated for con-comitant changes in SRF alternative splicing. RT-PCRconfirmed that in the first 24–48 hours of myogenicdifferentiation SRF∆5 isoform synthesis is graduallysuppressed, whereas SRF isoform production increas-es (Figure 5b). We next determined the effect ofmechanical stretch upon SRF isoform production inmouse lung cell and organ culture. Mechanical stretchelicited the same change in SRF isoform productionobserved during in vivo and in vitro SM myogenesis.Four hours of sustained uniaxial stretch caused sup-pression of SRF∆5 mRNA synthesis with a concomi-tant increase in SRF mRNA levels (Figure 5c). Consis-tent with these changes, after 12 hours in culturenonstretched cells synthesized SRF and SRF∆5 whilestretched cells synthesized only SRF (Figure 5c). A sim-ilar pattern of SRF isoform production in response todextran-induced peribronchial mesenchymal stretchwas seen in mouse lung organ cultures (Figure 5d).Mesenchymal cells isolated from human lungs at week16 of gestation also synthesized SRF and SRF∆5 but,upon spread- and stretch-induced SM differentiation,

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Figure 5(a) RT-PCR and immunoblot demonstrate the presence of SRF∆5in fresh (uncultured) mouse undifferentiated peribronchial mes-enchymal cells on day 11 (E11) and absence of SRF∆5 with con-comitant increment in SRF on day 14 (E14), after the peribronchialcells become SM cells. S18 represents an internal control. Theincrement in SRF isoforms was best seen at the protein rather thanat the message level. (b) RT-PCR shows SRF and SRF∆5 mRNAchanges along with cell spread–induced SM differentiation in cul-ture. Notice the increment in SRF mRNA and the decrease and dis-appearance of SRF∆5 mRNA. (c) RT-PCR demonstrates rapid dis-appearance of SRF∆5 mRNA and increments in SRF mRNA upon 4hours of sustained stretch. The same change is seen at the proteinlevel after 12 hours in the immunoblot in the lower panel. (d) Effectof peribronchial mesenchymal cell stretch on SRF and SRF∆5 iso-forms in lung organ cultures. One percent dextran inside the air-ways led to suppression of SRF∆5 and increase in SRF (lane 2),whereas dextran in the medium outside the lung explants main-tained the SRF isoform profile characteristic of undifferentiatedmesenchymal cells (lane 3). (e and f) Effect of cell spreading (e)and stretching (f) on SRF∆5 in human fetal mesenchymal cells.Notice that the very low levels of SRF∆5 mRNA found in the humancells are likely a reflection of their more advanced stage of SM dif-ferentiation. Results shown are representative of three experiments,each done on duplicate samples per treatment.

Figure 6(a) Transfection of SM-differentiating cells with SRF and SRF∆5expression vectors, empty vector, or nontransfected controls. Prima-ry cultures of mouse lung embryonic mesenchymal cells were platedunder conditions that allow cell spreading. The cells were transfect-ed 1 hour after attachment was completed. In the experiments inwhich the cells were stretched, transfection and stretching were ini-tiated concomitantly. The cells were lysed 18 hours after transfec-tion. SRF overexpression stimulates and SRF∆5 overexpressioninhibits SM differentiation. The vector alone does not affect differ-entiation (compare with nontransfected control cells). (b) SRF∆5overexpression inhibits stretch-induced SM differentiation. Noticethat, as expected, untransfected stretched cells synthesize higher lev-els of SRF than untransfected nonstretched ones (lane 1). Theseexperiments were repeated four times with similar results.

SRF∆5 isoform synthesis was suppressed whereas SRFisoform production increased (Figure 5, e and f). Thevery low levels of SRF∆5 found in these cells are likelya reflection of a more advanced stage of myogenesiscompared with the murine counterparts.

SRF stimulated and SRF∆5 inhibited spontaneous and stretch-induced myogenic differentiation. We first determined theeffect of overexpressing SRF and SRF∆5 isoforms inmouse embryonic mesenchymal cells undergoingspread-induced SM differentiation in culture. Theprocess of SM differentiation was either inhibited orfacilitated by transfecting these cells with SRF∆5 or SRF(Figure 6a). We then determined the effect of transfect-ing SRF∆5 into stretched cells. In these studies the cellswere transfected and stretched immediately uponattachment and were also allowed to spread to facilitatetransfection. These studies showed that stretch-inducedmyogenesis was blocked by expression of SRF∆5 (Figure6b). Metabolic radiolabeling demonstrated no differencein protein synthesis between transfected and untrans-fected cells. At the end of the culture period nontrans-fected cells incorporated [35S]-methionine at a rate of21,540 ± 410 cpm/µg protein, cells transfected with

empty vector incorporated 20,440 ± 520 cpm/µg protein,and cells transfected with SRF∆5 plasmid incorporated21,030 ± 560 cpm/µg protein. This indicated that theinhibitory effect of transfected SRF∆5 was not due to anonspecific decrease in protein synthesis.

Human hypoplastic lungs related to conditions that impairpulmonary stretch/distention showed poor visceral myogenesiswith a consequent decrease in elastin deposition. Immunos-taining with anti-SM α-actin demonstrated a severedecrease in the amount of visceral SM, includingbronchial and interstitial SM cells, in all of the humanhypoplastic lungs studied (Figure 7, arrows). The max-imal inhibition in visceral myogenesis was seen in areasof collapsed lung parenchyma resulting from diaphrag-matic hernia where there were almost no visceral SMcells (Figure 7c). Epithelial, endothelial, and vascularSM cells in the hypoplastic lungs did not show signifi-cant differences in amount and distribution comparedwith controls (Figure 7, d–g, and arrowheads for vas-cular SM). Immunohistochemical studies demonstrat-ed elastin deposition in the bronchi and bronchioli ofnormal fetal lungs (Figure 7h, arrows), whereashypoplastic fetal lungs of the same gestational age

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Figure 7Immunohistochemistry showing paucity of visceral SM cells in human fetal hypoplastic lungs. Shown are histological sections from normallung (a), hypoplastic lung caused by oligohydramnion (b), and hypoplastic lung caused by diaphragmatic hernia (c), all at 22 weeks of ges-tation, immunostained for SM α-actin. There is a significant decrease in bronchial and interstitial SM cells (arrows) in the hypoplastic lungs(b and c), particularly in those compressed by intrathoracic herniation of abdominal viscerae due to diaphragmatic hernia (c). The vascu-lar musculature seems unaffected (arrowheads). In the same hypoplastic lung shown in b, the epithelial cells, immunostained for low–molec-ular-weight cytokeratins (e), and the endothelial cells, immunostained for PECAM-1 (g), show no changes compared with controls (d andf). Photos (h and i) demonstrate immunohistochemistry showing decrease in tropoelastin deposition in human hypoplastic lungs. (h) His-tological sections from the normal lung at 20 weeks of gestation demonstrate tropoelastin deposition around bronchi and bronchioli andat scattered interstitial sites (arrows). (i) Histological sections from same age hypoplastic lung reveals essentially no tropoelastin deposi-tion, with the exception of vascular SM that shows no changes in tropoelastin when compared with controls (arrowheads). Bar, 60 µm ina–e and h and i. Bar, 100 µm in f and g. (j) RT-PCR and immunoblot show stretch-induced upregulation of tropoelastin expression in mouselung embryonic mesenchymal cells undergoing myogenic differentiation. (k) Immunoblot shows stretch-induced upregulation of tropoe-lastin synthesis in human lung embryonic mesenchymal cells undergoing myogenic differentiation. Results shown in j and k are representa-tive of three experiments conducted in duplicate sample per treatment.

showed no elastin deposition except for the blood ves-sels (Figure 7i, arrowheads). Mouse and human SM-dif-ferentiating cells subjected to stretch increased theirsynthesis of tropoelastin (Figure 7, j and k). These stud-ies suggested that the decrease in elastin seen inhypoplastic lungs may be the combined effect of fewercells producing less tropoelastin per cell.

SRF∆5 was present in hypoplastic lungs but not in normalage-matched controls. Intact RNA was isolated from fiveout of the nine hypoplastic lungs, ages 20 to 25 weeks(two samples of diaphragmatic hernia, both isolateralto the hernia, and three cases of oligohydramnion). Inaddition, RNA was isolated from five age-matched con-trols. RT-PCR amplified both SRF and SRF∆5 in thefive hypoplastic lungs, but only SRF isoform in the con-trols (Figure 8). Furthermore, the levels of SRF appearedhigher in the controls than in the hypoplastic lungs.

DiscussionStretch plays a critical role in initiating and maintaining lungvisceral myogenesis. Although the morphological aspectsof visceral myogenesis are well documented, the stimulithat initiate and control the process have remained elu-sive. Based on the hypothesis that mechanical stimuliare an important factor in SM development, we devisedcell and organ culture systems to determine the role ofstretch in bronchial myogenesis. These studies demon-strated that mechanical stretch alone is sufficient toinduce lung undifferentiated mesenchymal cells to fol-low a myogenic pathway. Similarly, SM-differentiatingcells responded to stretch by upregulating the expres-sion of SM proteins. The critical contribution of cellstretching to visceral myogenesis was best exemplifiedin mouse lung organ cultures, where bronchial SM dif-ferentiation was either induced or prevented by modu-lating the airway intraluminal pressure.

Fully differentiated SM cells and embryonic mes-enchymal cells from gut or kidney did not respond tostretch under the conditions studied. These observa-tions suggest a lower susceptibility to mechanical stim-uli. Interestingly, in a previous study we found thatembryonic mesenchymal cells from lung, gut, and kid-ney equally differentiated into SM upon spreading (11).The reasons for this unexpected difference in responseto stretch are unclear. However, they may be related tothe unique type of mechanical stimuli sustained by thelung. Besides the stretch generated by intraluminalhydrostatic pressure common to all three organs, thelung also sustains the rhythmic stretch produced byintrauterine breathing. Therefore, it could be possiblethat lung mesenchymal cells are highly dependent onstretch as the main stimulus for muscle differentiation,while additional factors are required in the intestineand urinary tract.

The most extensively reported effect of cell stretchingis the stimulation of cell proliferation (22, 23). Howev-er, we found that induction of myogenesis by cellstretching was independent of cell growth, as it tookplace despite serum deprivation. In this regard, our pre-

vious studies demonstrated that SM myogenesisinduced by cell spreading/elongation is also independ-ent of cell growth (10, 11).

Stretch induces a change in SRF RNA alternative splicingthat promotes SM-specific gene expression. Using these cul-ture systems, we began to explore the molecular mech-anisms underlying stretch-induced myogenesis. Wefocused initially on SRF because this transcription fac-tor stimulates expression of a broad range of SM-spe-cific proteins. The biological activity of SRF was there-fore compatible with the effects of stretch. It has beenshown recently that SRF has three alternative splicedvariants in addition to the full SRF molecule (17, 18).We found that one of them, SRF∆5 (lacking exon 5), issynthesized by lung undifferentiated mesenchymalcells, which also express relatively high levels of SRF.Our studies demonstrated that during the first stagesof myogenesis, SRF∆5 synthesis is suppressed in favorof increased SRF production. This change in SRF iso-form profile seems to play an important functional rolein stimulating SM differentiation, as indicated by thetransfection studies. In this regard, it should bestressed that since these were done on primary cultures,without further selection for transfected cells, theresults reflect a cell pool that also included nontrans-fected cells. Therefore the differences in SM proteinexpression are likely to be greater than those detected.

Our findings correlated well with those of Belaguli etal. (17). Using promoter-reporter constructs, this groupshowed that SRF∆5 behaves as a powerful dominant-negative isoform inhibiting SM α-actin and SM22expression elicited by SRF. However, SRF∆5 can alsostimulate muscle-specific gene expression, dependingupon the cell type as well as other factors (18).

SRF∆5 binds to SRF and to DNA-SRF elements (17).Therefore, it has been proposed that the repression ofSM protein expression by SRF∆5 is due to the formationof SRF-SRF∆5 heterodimers as well as the interaction ofSRF∆5 with SRF promoter-binding sites (17). These pos-sibilities seem to be refuted by the fact that SRF is pres-ent in lung undifferentiated mesenchymal cells in sig-nificantly higher levels than SRF∆5. However, SRF∆5

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Figure 8 RT-PCR shows SRF∆5 and SRF isoforms in five human hypoplasticlungs: lane 1, lung isolateral to diaphragmatic hernia, week 20; lane2, lung isolateral to diaphragmatic hernia, week 22–23; lane 3, lungfrom oligohydramnion, week 25; lane 4, lung from oligohydramnion,week 21; lane 5, lung from oligohydramnion, week 24. Lanes 6–11show lungs from five matching controls with the presence of higherSRF levels and absence of SRF∆5. Lane 11 represents SRF isoformsin day-11 mouse lung, and it has been used as an additional control.

may function only as a selective antagonist of SRF myo-genic activity without participation in other SRF-medi-ated processes. In such a case the small amount ofSRF∆5 could be recruited to few specific DNA sites bymeans of their comparatively high binding affinity toSRF∆5 or through binding to their associated proteins.

SRF∆5 mRNA has been identified in adult aorta andstriated muscle (17, 18). This may suggest that otherSRF isoforms could substitute for SRF∆5 antimyo-genic function, depending on the type of muscle(smooth or striated), or even more, that myogenesis indifferent sites proceeds through different pathways.Alternatively, SRF∆5 may be re-expressed in some mus-cles after their differentiation is completed, perhaps toserve other functions. Supporting the last possibility,SRF∆5 mRNA has been identified in lysates of adultorgans bearing SM, including stomach (17) and uterus(18), but was not detected in adult intestinal SM (Y.Yang and L. Schuger, unpublished observation).

Presence of SRF∆5 and paucity of bronchial and interstitialSM cells in human hypoplastic lungs suggest that visceral myo-genesis may be impaired by decreased stretching. Lunghypoplasia is a severe and often fatal neonatal condi-tion characterized by a low lung-to-body weight ratio(small lungs) (21). The two main causes of lunghypoplasia are diaphragmatic hernia and oligohy-dramnion (21). Although the pathogenesis of lunghypoplasia associated with these two conditions is notwell understood, low mechanical tension is thought toplay an essential role. The developing lung is filledwith liquid, mainly amniotic fluid but also fluidsecreted by the lung itself (13, 21). In oligohydramnionthe amniotic fluid volume is diminished, producing adecrease in intrapulmonary hydrostatic pressure (24,25). In diaphragmatic hernia the abdominal organs aredisplaced into the thoracic cavity, thereby precludingnormal lung expansion and indirectly decreasing theairway hydrostatic pressure (26, 27). Diminished intra-pulmonary pressure and impaired distention result inless mechanical stretch (24–30), inasmuch as correct-ing the latter in utero by artificially increasing the air-way hydrostatic pressure ameliorates these two typesof lung hypoplasia (26, 29, 30).

Although no bronchial or interstitial SM abnormali-ties have been reported in hypoplastic lungs, our exper-imental data suggested that they may be defective.Indeed, immunohistochemistry demonstrated a severedecrease in bronchial as well as interstitial SM cells inhuman hypoplastic lungs compared with age-matchedcontrols and to the other main cell types in the lung.The normal development of vascular SM in hypoplas-tic lungs was consistent with our in vitro observations.If mechanical stretching also induces vascular myoge-nesis, then the stretching forces would have to comefrom the hydrostatic pressure within blood vessels,which is not decreased in hypoplastic lungs.

Elastin represents a critical structural and function-al component of the lung. Since tropoelastin, themonomeric form of elastin, is synthesized mainly by

SM cells (6, 31), we determined the deposition of thisextracellular matrix constituent in hypoplastic lungs.Consistent with the lack of SM cells, immunohisto-chemical studies indicated that hypoplastic lungs con-tain significantly less peribronchial and interstitialtropoelastin than controls. Absence of elastin was alsoreported in an ultrastructural study of human oligo-hydramnion-related hypoplastic lungs (32). In addi-tion, our studies suggested that stretch induces elastindeposition not only by promoting SM differentiation,but also by stimulating tropoelastin synthesis by dif-ferentiating SM myoblasts. Therefore, the decreasedelastin levels found in hypoplastic lungs may be thecombined effect of fewer SM cells and less tropoelastinproduction by each of them.

Our studies demonstrated the presence of SRF∆5and relatively lower levels of SRF in hypoplastic lungscompared with age-matched controls. These findingsindicated an abnormal pattern of SRF splicing in thehypoplastic lungs and lent additional support to therole of mechanical stimuli in visceral myogenesis. Inaddition, the higher level of SRF∆5 in the hypoplas-tic lungs compared with the embryonic mouse sug-gested that this isoform might be more prevalent inthe early human lung than in its murine counterpart.The signaling pathways connecting cell stretch toSRF alternative splicing are currently unknown. Like-wise there is no information on other factors thatmay regulate SRF isoform profile in addition tomechanical stimuli. Therefore, it is presently unclearhow specifically SRF-splicing regulation is associat-ed with the latter. In fact, the possibility exists thatabnormalities in SRF RNA alternative splicing repre-sent a common denominator for diseases character-ized by abnormal SM or SM-related cells in the lung,such as chronic lung fibrosis, asthma, or lymphangi-oleiomyomatosis. Further studies are required toaddress this possibility.

AcknowledgmentsThis work has been supported by National Heart,Lung, and Blood Institute grant HL-48730.

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